ABSTRACT To clarify distinct genetic profiles of colorectal cancers based on tumor location (left- and right-sided), we evaluated the status of loss of heterozygosity (LOH), CpG islands methylation phenotype (CIMP), microsatellite instability (MSI), and mutations of p53, Ki-ras, and APC genes in 119 colorectal cancers. Statuses of LOH (at 5q, 8p, 17p, 18q, and 22q), MSI, and CIMP (MINT1, MINT2, MINT31, MLH-1, MGMT, p14, p16, and RASSF1A) were determined using microsatellite polymerase chain reaction and methylation-specific polymerase chain reaction coupled with a crypt isolation method, respectively. In addition, mutations of p53, Ki-ras, and APC genes were also examined. LOH, MSI, and CIMP status allowed us to classify samples into two groups: low or negative and high or positive. Whereas the frequency of p53 mutations in the LOH-high status was significantly higher in left-sided cancers than in right-sided cancers, CIMP-high in the LOH-high status and MSI-positive status were more frequently found in right-sided cancers compared with left-sided cancers. Finally, location-specific methylated loci were seen in colorectal cancers: type I (dominant in right-sided cancer) and type II (common in both segments of cancer). Our data confirm that distinct molecular pathways to colorectal cancer dominate in the left and right sides of the bowel.

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Purpose The findings from epidemiologic studies of dyslipidemia and colorectal cancer risk have been conflicting. We performed a dose–response meta-analysis of published prospective studies to assess the aforementioned association. Methods Relevant studies that reported the association between the components of dyslipidemia (serum triglyceride, total cholesterol, and high-/low-density lipoprotein cholesterol) and colorectal cancer risk were identified by searching PubMed until the end of May 2014. We pooled the relative risks (RRs) from individual studies using a random- and fixed-effects models and performed dose–response, heterogeneity, and publication bias analyses. Results Seventeen prospective studies, including 1,987,753 individuals with 10,876 colorectal cancer events, were included in the meta-analysis. The overall pooled RR for high versus low concentrations for triglyceride (n = 9 studies) was 1.18 (95 % CI 1.04–1.34; I 2 = 47.8 %), for total cholesterol (n = 10 studies) was 1.11 (95 % CI 1.01–1.21; I 2 = 46.7 %), for high-density lipoprotein cholesterol (n = 6 studies) was 0.84 (95 % CI 0.69–1.02; I 2 = 42.5 %), and for low-density lipoprotein cholesterol (n = 3 studies) was 1.04 (95 % CI 0.60–1.81; I 2 = 82.7 %). In the dose–response analysis, the overall pooled RR was 1.01 (95 % CI 1.00–1.03; I 2 = 0 %) per 50 mg/dL of triglyceride and 1.01 (95 % CI 0.97–1.05; I 2 = 64.3 %) per 100 mg/dL of total cholesterol. Conclusions This meta-analysis of prospective studies suggests that dyslipidemia, especially high levels of serum triglyceride and total cholesterol, is associated with an increased risk of colorectal cancer, whereas high-density lipoprotein cholesterol might associate with a decreased risk of colorectal cancer. Further studies are warranted to determine whether altering the concentrations of these metabolic variables may reduce colorectal cancer risk.

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BackgroundA large proportion of US Medicare beneficiaries undergo earlier-than-recommended follow-up colonoscopies after negative screening colonoscopy. Such practice entails substantial cost and added risk.AimsTo compare the risk of colorectal cancer (CRC) associated with varying follow-up colonoscopy intervals following a negative colonoscopy, and to determine whether the potential benefit of a shorter colonoscopy follow-up interval would differ by gender.Methods
We conducted a weighted cohort study using the Surveillance, Epidemiology and End Results-Medicare linked database (1991–2006) among 932 370 Medicare enrollees who are representative of the entire US elderly population. We compared the cumulative incidence of CRC among patients who underwent follow-up colonoscopies at different intervals following a negative colonoscopy. The primary outcome was incident CRC.ResultsThe eligible study cohort (n = 480 864) included 106 924 patients who underwent ≥1 colonoscopy. Men were more likely to require polypectomy during their initial colonoscopy than women. Compared to the recommended 9–10 year follow-up colonoscopy interval, an interval of 5–6 years was associated with the largest CRC cumulative risk reduction [i.e. 0.17% (95% CI: 0.009–0.32%)]. The magnitude of risk reduction associated with shorter colonoscopy follow-up intervals was not significantly different between men and women.Conclusions
Among elderly individuals who undergo a negative colonoscopy, the magnitude of reduction in the cumulative CRC risk afforded by earlier-than-recommended follow-up colonoscopy is quite small, and probably cannot justify the risk and cost of increased colonoscopy frequency. In addition, there are insufficient differences between men and women to warrant gender-specific recommendations.

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Upon binding their cognate receptors, ERα (ESR1) and ERβ (ESR2), estrogens activate intracellular signaling cascades that have important consequences for cellular behavior. Historically linked to carcinogenesis in reproductive organs, estrogens have also been implicated in the pathogenesis of different cancer types of non-reproductive tissues including the colon. ERβ is the predominant estrogen receptor expressed in both normal and malignant colonic epithelium. However, during colon cancer progression ERβ expression is lost, suggesting estrogen signaling may play a role in disease progression. Estrogens may in fact exert an anti-tumor effect through selective activation of pro-apoptotic signaling mediated by ERβ, inhibition of inflammatory signals and modulation of the tumor microenvironment. In this review we analyze the estrogen pathway as a possible therapeutic avenue in colorectal cancer, we report the most recent experimental evidence to explain the cellular and molecular mechanisms of estrogen-mediated protection against colorectal tumorigenesis, and we discuss future challenges and potential avenues for targeted therapy.

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Western countries, especially Australia and Europe.6,10For example, MSI-positive cancers are preferentiallyfound in right-sided colon cancer and in older women.5,6Hawkins et al7indicated a higher incidence of CIMP-positive tumors in right-sided colon cancer comparedwith left-sided colon cancers. On the other hand, CIN issaid to characterize left-sided colorectal cancers.10These findings have provided further evidence for theexistence of at least two mechanisms of colorectal can-cer.6One group (CIMP?/MSI?) occurs predominantly inthe right-sided colon and the other (CIN) in the left-sidedcolon.6,10However, the underlying molecular features arelikely to show considerable overlap between the two can-cers.6Therefore, it is important to study genetic colorec-tal carcinogenesis while taking into account whether thetumor is located in the left- or right-sided colon.In this study, we examined sporadic colorectal can-cers in an attempt to determine their genetic profiles interms of LOH status, MSI, CIMP, and tumor location.Mutations of p53, Ki-ras, and APC genes that are asso-ciated with LOH-high status (which occurs with almostthe same frequency as CIN) were evaluated to clarifytheir relationships to the genetic profiles at the two sites.Materials and MethodsA total of 119 primary colorectal cancers and corre-sponding normal tissue specimens were obtained frompatients at the Iwate Medical University School of Medi-cine. Pathological diagnosis and staging were performedaccording to a combination of a Japanese classificationand the modified Dukes’ classification.11,12Tumor loca-tions were noted as left- or right-sided.DNA ExtractionCrypt isolation from the tumor and normal mucosa wasperformed in accordance with a previously reportedmethod to obtain pure glands.13,14The isolated glandwas processed routinely to confirm its nature using par-affin-embedded histological sections. Contamination byother materials such as interstitial cells was not evident inthe samples that were examined, as described in previ-ous reports.15,16DNA from the tumor and from corre-sponding normal crypts was extracted by standard so-dium dodecyl sulfate proteinase K treatment.Microsatellite AnalysisLOH studies were performed by polymerase chain reac-tion (PCR) amplification of 13 highly polymorphic micro-satellite markers (D5S107, D5S346, D5S299, D5S82,D8S201, D8S513, D8S532, TP53, D18S487, D18S34,D22S274, D22S1140, and D22S1168) located at fivechromosomal loci (5q, 17p, 18q, 8p, and 22q).17Micro-satellite sequences were obtained from specific primersreported in the GDB Human Genome Database (http://gdbwww.gdb.org/gdb/). PCR amplification was per-formed in a 25-?l reaction volume, containing approxi-mately 10 ng of genomic DNA, 1 ?mol/L of each primer,0.2 mmol/L deoxynucleotide triphosphate, 1? reactionbuffer containing 1.5 mmol/L MgCl2, and 1.5 U of Taqpolymerase (Boehringer Mannheim Co., Mannheim, Ger-many). Samples were processed for 25 to 30 cycles, witheach cycle consisting of 30 seconds at 94°C, 1 minute at55 to 58°C, and 2 minutes at 72°C, followed by a final10-minute extension at 72°C. PCR products were loadedonto 6% polyacrylamide gels and run on an ABI PRISM377 DNA Sequencer (Applied Biosystems, Foster City,CA). The data were collected automatically and analyzedby GeneScan 3.1 software (Applied Biosystems). LOHwas determined by calculating the ratio of the peak areasof the constitutional alleles, as described previously.16Inthis study, we defined LOH as more than a 50% differ-ence in this ratio.Scoring of LOH StatusLOH status was scored according to following criteria. Atumor sample was considered to be LOH-high if two ormore of the markers showed LOH. When one or none ofthe markers showed LOH, the tumor was designated asLOH-low.Analysis of MSIThe primers proposed by the National Cancer InstituteWorkshop on Microsatellite Instability (BAT25, BAT26,D5S346, D2S123, and D17S250) were used in thisstudy.18Products were run on an ABI PRISM 377 fluo-rescent DNA sequencer. PCR conditions are describedelsewhere.16MSI was defined as the presence of anadditional peak. A tumor sample was considered to beMSI-high (MSI-H) when two or more of the markers dem-onstrated instability and MSI-low when only one markerwas unstable. However, tumors showing one alterationusing the above criteria and categorized as MSI-low wereconsidered MSI-negative or MSS in this analysis.Mutation Analysis of the p53, APC, and Ki-rasGenesSequencing of PCR-amplified products was used to de-tect mutations of exons 5 to 8 of the p53 gene, exon 1 ofthe Ki-ras gene, and the mutation cluster region of theAPC gene in patients’ normal mucosa and tumor DNAsamples. PCR conditions and sequencing of mutationswere performed as described previously.15,17Direct se-quencing was performed using fluorescently labeleddideoxynucleotide triphosphates by automated DNA se-quence analysis (373A sequencer; Applied Biosystems).CIMP of CarcinomasCIMP status was determined for carcinomas, which wereevaluated at eight loci (MINT-1, MINT-2, MINT-31, p14,p16, MGMT, MLH-1, and RASSF-1A) after bisufite treat-ment. These loci are frequently methylated in colorectal194JMD May 2006, Vol. 8, No. 2Sugai et al

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carcinomas.7,9Methylation-specific PCR was performedusing specific primers for either the methylated or theunmethylated examined primers. Primers and conditionshave been described previously.19,20In vitro methylatedDNA was used as a positive control for methylation, andwater was used as a negative control. The results ofmethylation-specific PCR were scored when there was aclearly visible band on the gel after PCR with methylatedand unmethylated primers. Electrophoresis results wereinterpreted by two independent investigators. When adiscrepancy between the two was found, a third opinionwas sought. Carcinomas were classified as CIMP-nega-tive/low if less than three loci were methylated and CIMP-high if more than two loci were methylated. Alternatively,CIMP-negative/low and -high were classified into CIMP-low and -high, respectively.Statistical AnalysisThe data were analyzed using the ?2test with the aid ofStatView-IV software (Abacus Concepts, Berkeley, CA).Samples were determined to be significantly different atP ? 0.05.ResultsIn the present study, differences in clinicopathologicalfindings and genetic alterations in left-sided and right-sided colorectal carcinomas were analyzed. Molecularprofiles were categorized into four types: LOH-high, low-LOH, MSI, and CIMP. Clinicopathological findings for left-and right-sided colorectal cancers are listed in Table 1.Molecular Alterations in Left- and Right-SidedCancersAlthough the frequency of LOH-high in left-sided colorec-tal cancer (72 of 84 cases, 85.7%) was higher than that ofright-sided cancer (24 of 35 cases, 68.6%), this differ-ence was not statistically significant. There was a signif-icant difference in the frequencies of p53 mutations foundin left-sided (49 of 84, 57.6%) and right-sided (9 of 35,25.7%) cancers (P ? 0.05). However, no differenceswere found in the frequencies of Ki-ras and APC muta-tions. The CIMP-high status was found in 38 of 119 colo-rectal cancers that were examined (31.9%). The CIMP-high status was more common in right-sided cancers (22of 35, 62.9%) compared with left-sided cancers (21 of 84,25%) (P ? 0.01).Mutations of p53, Ki-ras, and APC Genes andCpG Islands Methylation Phenotype in LOH-High StatusThe frequency of p53 mutations in LOH-high statuscancers was significantly higher for left-side cancersthan for right-side cancers (Figure 1a, P ? 0.01). Incontrast, no significant differences in the frequenciesof Ki-ras and APC mutations in LOH-high status can-cers were observed for left- versus right-sided cancers(Table 2). On the other hand, the CIMP-high status inLOH-high cancers was statistically higher in right-sided (12 of 24, 50%) than in left-sided colon cancers(14 of 72, 19.4%) (Figure 1a, P ? 0.01). In addition,there were no differences in the frequency of LOH ateach chromosomal locus between left-sided and right-sided cancers, as shown in Table 2.Mutations of p53, Ki-ras, and APC Genes andCpG Islands Methylation Phenotype in LOH-Low StatusThe frequency of LOH-low status was 18 of 119(15.1%) in our study. Although no p53 mutations weredetected in right-sided colorectal cancers with LOH-low status (0 of 7), no significant difference was foundcompared with the frequency of p53 mutations in theleft-sided cancers (3 of 11, 27.3%). Ki-ras and APCgene mutations in the LOH-low status were found morefrequently in right-sided colorectal cancers (5 of 7,71.4%; and 3 of 7, 42.9%, respectively) compared withleft-sided cancers (4 of 11, 36.4%; and 1 of 11, 9.1%,respectively), but again these differences did notreach statistical significance. The frequency of CIMPstatus in the LOH-low cancers was high for left-sidedcancers (6 of 11, 54.5%) as well as for right-sidedcancers (6 of 7, 85.7%). These findings are shown inFigure 1b.Mutations of p53, Ki-ras, and APC Genes andLOH-High Status in CpG Islands MethylationPhenotype Excluding MSI TumorsThe frequencies of CIMP-high status were significantlyhigher in right-sided cancers (20 of 84, 24.7%) than inleft-sided cancers (18 of 35, 51.4%) (P ? 0.01). Althoughp53 gene mutations in the CIMP-high status cancerswere found more frequently in left-sided (10 of 20, 50%)Table 1.Clinicopathological Findings between Left- andRight-Sided Colorectal CancersLeft-sided (%) Right-sided (%)TotalSex (male/female)Age (mean)Histological typeWDAMDAPDAMCStageABCD84 (70.6)56/2846–93 (64.6)35 (29.4)20/1522–94 (62.8)18 (21.4)65 (77.4)1 (1.2)06 (17.1)22 (62.9)2 (5.7)5 (14.3)14 (16.7)33 (39.3)22 (26.2)15 (17.9)4 (11.4)16 (45.7)10 (28.6)5 (14.3)WDA, well-differentiated adenocarcinoma; MDA, moderately differ-entiated adenocarcinoma; PDA, poorly differentiated adenocarcinoma;MC, mucinous carcinoma.Molecular Alterations of Colorectal Cancers 195JMD May 2006, Vol. 8, No. 2

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compared with right-sided colorectal cancers (3 of 18,16.7%), the difference did not reach a statistically signif-icant level, as shown in Figure 1c (P ? 0.068). On theother hand, although the frequency of Ki-ras mutationswas relatively higher in right-sided cancers (12 of 18,66.7%) than in left-sided cancers (8 of 20, 40%), nosignificant difference between the two sites was found.There was also no significant difference in the frequencyof APC gene mutations in the CIMP-high status cancersbetween the two locations (left-sided, 8 of 20, 40%; right-sided, 7 of 18, 38.9%).The overall frequencies of promoter hypermethylationin right-side cancers were higher, with the exception ofMINT1, MGMT, and RASSF-1A. In particular, the frequen-cies of MINT-2, MLH-1, p14, and p16 hypermethylationsin right-sided cancers were significantly higher than forleft-sided cancers (P ? 0.01, P ? 0.05, P ? 0.01, and P ?0.01, respectively). The differences for MINT31 did notreach a significant level (P ? 0.052). The data are shownin Figure 2. In the present study, among the eight CpGislands occurring within known promoter regions in colo-rectal cancers, the hypermethylation patterns fell into twotypes. One type was found to be frequently methylated inright-sided colon cancers (type I). The other type wasmethylated in both types of colorectal cancers (type II).Whereas MINT-2, MLH-1, p14, and p16 methylationswere classified as type I, MINT-1, MGMT, and RASSF-1Amethylations were grouped into type II.p53, Ki-ras, and APC Gene Mutations, CIMP,and LOH Status in MSI-Positive StatusA significant difference in the frequency of MSI betweenleft- and right-sided colorectal cancers was found (1 of84 cases vs. 4 of 35 cases, P ? 0.05). In the presentstudy, all MSI-positive cases that were observed had aCIMP-high status. Mutations of Ki-ras and APC geneswere found in one left-sided cancer. In addition, a p53mutation was detected in one right-sided cancer. TheTable 2.Frequencies of Allelic Imbalances at Cancer-RelatedChromosomal Loci in Left- and Right-SidedColorectal CancersTotal (left/right)Left-sided(%)Right-sided(%)17p5q18q8p22q84/3584/3584/3584/3584/3554/77 (70.1)51/83 (61.4)69/79 (87.3)51/81 (60.7)42/81 (51.9)15/25 (60)16/30 (53.3)24/28 (85.7)16/30 (53.3)15/29 (51.7)Figure 1. a: Relationship of mutations in p53, Ki-ras, and APC in LOH-high status tumors based on tumor location. b: Relationship of mutations in p53, Ki-ras,and APC in LOH-low status tumors based on tumor location. c: Relationship of mutations in p53, Ki-ras, and APC in CIMP-high status tumors based on tumorlocation.Figure 2. Frequencies of methylation of MINT1, MINT2, MINT31, MLH-1,MGMT, p14, p16, and RASSF1A promoters for left- and right-sided cancers.196JMD May 2006, Vol. 8, No. 2Sugai et al

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other three MSI-positive tumors showed no mutations.LOH-status could not be confirmed because of the pres-ence of MSI-positive at individual chromosomal loci. Fi-nally, representative examples of molecular analysis ofleft- and right-sided cancers are shown in Figures 3and 4.Tumor Mutation Spectra of p53, Ki-ras, andAPC Mutations in Left- and Right-SidedCancersAlthough the proposed sequence of genetic alterationsleading to the development of colorectal cancer involvesmutations in all three genes (p53, Ki-ras, and APC), asignificant percentage of the tumors contained mutationsin only one of these genes: p53 (22 of 119, 18.5%), Ki-ras(18 of 119, 15.1%), and APC (11 of 119, 9.3%). In ourstudy, we characterized tumor mutation spectra in left-and right-sided cancers. The detailed results are shownin Table 3. Only 8 (9.5%) and 2 (5.7%) tumors of left- andright-sided cancers, respectively, contained mutations inall 3 genes, whereas 12 tumors (14.3%) of left-sided and6 (8.6%) of right-sided cancers contained no mutations.The most common combination of mutations in left-sidedtumors was p53 and APC (14 of 84, 16.7%), whereas thatof right-sided tumors was Ki-ras and APC mutations (4 of35, 11.4%).DiscussionIn this study, analysis of LOH and MSI status was per-formed for 119 colorectal cancers to assess the possibledifferences between left- and right-sided colorectal can-cers. Although we did not observe significant differencesin LOH status between the two locations, LOH-high statuswas found in 84.5% of left-sided colorectal cancers and68.6% of right-sided colorectal cancers. In addition, weobserved a tendency toward a similar frequency of LOHat each individual locus in the two segments of colorectalcancers. On the other hand, the frequency of MSI wassignificantly higher in right-sided colorectal cancers thanin left-sided colorectal cancers.7,21However, MSI-posi-tive cancer is not generally representative of right-sidedcolorectal cancers, given that it was found in only 11.4%of right-sided colorectal cancers in our study. We notethat the remainder, approximately 90%, of right-sidedcolorectal cancers consisted of MSS-type tumors, espe-cially LOH-high-type tumors (approximately 70% of right-sided cancers). These findings suggest that LOH-highstatus is a common genetic event in both segments ofcolorectal cancers and that the MSI type is a minor ge-netic type in right-sided cancers.In the present study, mutations in the p53 gene werepredominantly present in left-sided carcinomas. Theoverall percentage of p53 mutated tumors and the higherincidence of p53 mutations in left-sided carcinomas isFigure 3. A representative example of molecular alterations in left-sided cancer. b, d, f, h, and i: Multiple LOHs were seen (arrowhead). Although methylationat MINT 1 was found, MINT2, MINT31, p16, p14 (not shown), and RASSF1A promoters were not methylated. Furthermore, mutations of p53, Ki-ras, and APC geneswere found (arrowhead). k: A GGC to GAC transition in Ki-ras codon 12 was found, resulting in a Gly to Asp substitution (sequence by reverse primer). l: ACTG to CAG transversion in exon 5 of p53 gene was observed. m: Finally, a CGA to TGA transition in codon 805 of APC gene was found, resulting in a stop codon.The three detectable mutated nucleotides in tumor DNA are indicated by arrows in the photograph (k, l, and m).Molecular Alterations of Colorectal Cancers197JMD May 2006, Vol. 8, No. 2